Multiple lines of evidence support a critical role of infections in the etiology of secondary lesions that initiate childhood leukemia. This led to the hypothesis that delayed exposure to common infections predisposes to childhood pre-B ALL. Early exposure to infectious pathogens, e.g. through daycare attendance or vaccinations, leads to mild and typically subclinical immune responses and is associated with a significantly diminished risk to develop childhood ALL. By contrast, delayed infections are often more serious, cause more vigorous immune responses and predispose to childhood pre-B ALL. The central goal of this proposal is to experimentally test the 'delayed infections' hypothesis and to delineate mechanisms of genetic vulnerability of human pre-B cells in the context of infection. Pre-B cells undergo Rag1/2-dependent immunoglobulin V(D)J gene recombination and represent the cell of origin of childhood ALL. Rag1/2-mediated recombinase activity causes DNA double strand breaks and is associated with a low risk to acquire chromosomal translocations. This risk, however, dramatically increases when Rag1/2 enzymes are expressed concomitantly with the B cell-specific mutator enzyme AID. Hence, expression of Rag1/2 and AID is mutually exclusive and temporally separated in early and late B cell development, respectively: Rag1 and Rag2 expression is limited to immunoglobulin V(D)J gene recombination in pro- and pre-B cells. Conversely, AID expression is thought to be restricted to somatic hypermutation and class-switch recombination in mature B cells that have encountered antigen. In preliminary work for this proposal, we found that IL7R?/Stat5/Akt signaling is critical to keep normal pre-B cells in an antigen- unresponsive state. Upon withdrawal of IL7 or conditional deletion of Stat5, pre-B cells become fully responsive to antigen (e.g. LPS) and express AID at similar levels as mature B cells upon LPS stimulation. While high levels of IL7 in the bone marrow secure pre-B cells in an antigen-unresponsive state, we discovered a window of vulnerability during normal pre-B cell differentiation: Pre-B cell receptor signaling downregulates IL7R? expression and induces Rag1/2-mediated immunoglobulin light chain gene recombination in small resting (Fraction D) pre-B cells. Upon antigen encounter, Fraction D pre-B cells express high levels of AID in addition to Rag1/2, which dramatically increases their propensity to chromosomal translocations. Based on these findings, we hypothesize that Fraction D pre-B cells are highly susceptible to genetic lesions in the context of infection. We propose the following three Aims to test the prediction that the size of the Fraction D pre-B cell pool and the frequency and intensity of immune responses to infections will determine the likelihood of a pre-leukemic (e.g. TEL-AML1) pre-B cell clone to acquire critical secondary lesions.